MITOCHONDRIA AND APOPTOSIS (GM60554) John C. Reed, MD,PhD [Principal Investigator] Mitochondria play important roles in apoptosis and cell death. These energy-producing organelles are a major target of regulation by Bcl-2-family proteins, pro-apoptotic and anti-apoptotic proteins that integrate into mitochondria! membranes. During the previous funding period, we identified proteins that interact with and modulate the activity of Bcl-2-family proteins through novel (BH3-independent) mechanisms. These proteins include TR3 (Nur77), an orphan member of the retinoid/steroid family of transcription factors, which promotes apoptosis. Preliminary data suggest that TR3 interacts in a unique way with Bcl-2 and certain other anti-apoptotic Bcl-2-family members, converting them from apoptosis-suppressors to apoptosis-inducers. We call this class of proteins, Converters, because they convert Bcl-2 and Bcl-XL from anti-apoptotic to pro- apoptotic molecules. In our continuing efforts to understand mechanisms regulating the functions of Bcl-2-family proteins, we propose to investigate the Converter protein TR3, defining the molecular mechanisms involved and exploring the physiological significance. The central hypothesis we will test is that Converter proteins such as TR3 define a novel (non-BH3-based) mechanism for modulating the activity of Bcl-2-family proteins. Experiments are described that will address the issues of: (1) What controls the interactions of Converter protein TR3 with Bcl-2-family targets?; (2) How does Converter protein TR3 switch the phenotype of Bcl-2 and anti-apoptotic Bcl-2-family proteins it binds from protector to killer?; (2) What are the structural features of the unique interaction of Bcl-2 with TR3? (3) Under what circumstances is TRS's interaction with Bcl-2 physiologically important? Because dysregulation of mitochondria-dependent cell death pathways makes major contributions to illnesses where cell death is either insufficient (e.g. cancer, autoimmunity) or excessive (e.g., myocardial infarction, stroke; neurodegeneration), the insights gained from these investigations could find broad applicability to the improved treatment of several human diseases, particularly cancer where over-expression of Bcl-2 could potentially be converted from an advantage for tumors to a disadvantage, by exploiting Convert protein mechanisms.